A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be important to gain a detailed understanding of the impact of malaria on the generation and maintenance of immunological memory, the basis of all vaccines. This project represents a collaborative effort between Dr. Pierce, Dr. Louis Miller, Dr. Peter Crompton and with scientists at the Malaria Research and Training Center (MRTC) at the University of Bamako. Our goal is to gain an understanding of the generation, maintenance and activation of immunological memory in response to natural malaria infection. We determined the acquisition of both Pf-specific memory B cells (MBCs) and Pf-specific Abs using a proteome chip containing approximately 25% of the Pf proteome in a cohort of children and young adults in Kambila, Mali exposed to intense, seasonal Pf transmission. We documented that both the long-lived Pf-specific Abs and MBCs increased slowly, year by year, in a step-wise fashion. One focus of our future work is to understand what cellular and molecular mechanisms interfere with the rapid acquisition of protective immunity. We also identified an antibody signature associated with malaria immunity. Antibodies specific for a subset of 49 Pf proteins were found to be significantly increased in immune children as compared to nonimmune children. The increased antibody responses specific for the top five of these 49 proteins proved to be a highly sensitive and specific indicator of malaria immunity. A long term goal is to determine which of the Pf proteins, if any, could confer protection as a vaccine. Although classical antigen-specific MBCs were generated in response to malaria infections we also observed a large increase in the number of atypical FcRL4+ MBCs. Atypical FcRL4+ MBCs appear to be functionally impaired and activated only poorly through their B cell receptor (BCR). Atypical FcRL4+ MBCs were first identified in large numbers in the peripheral blood of HIV-infected viremic individuals suggesting that FcRL4+ MBCs may differentiate in response to chronic antigenic stimulation. We subsequently found that FcRL4+ MBCs were greatly expanded in individuals in our Malian cohort and could represent up to 30% of B cells in Malian children as young as two years of age. We subsequently showed FcRL4+ atypical MBC were increased in Peruvian adults exposed to low Pf transmission, but not to the extent observed in Malian adults (Peru mean 5.4%; Mali mean 13.1%) suggesting a correlation between Pf exposure and the generation of atypical FcRL4+ MBCs. In HIV viremic individuals following anti-retroviral therapy, the drop in HIV levels correlated with a drop in the number of atypical FcRL4+ MBCs, further suggesting that HIV drives the generation and/or maintenance of atypical FcRL4+ MBCs. Similarly we observed that children in rural Kenya exposed to ongoing Pf transmission had larger numbers of atypical FcRL4+ MBCs as compared to age-matched controls living in similar conditions but in an area where Pf transmission ceased five years earlier, suggesting that Pf drives the generation or maintenance of atypical FcRL4+ MBCs. We would like to better characterize the response of atypical FcRL4+ MBCs to antigenic stimulation and focused first on the impact of the inhibitory receptors that are uniquely expressed on atypical FcRL4+ MBCs on BCR signaling. We collaborated with Dr. Susan Moir and showed that siRNA knockdown of FcRL4 and Siglec-6 increased the proliferation of atypical FcRL4+ MBCs to HIV suggesting that FcRL4 and Siglec-6 dampen BCR signaling. Consistent with this observation we showed that expression of FcRL4 in human B cell lines disrupted BCR signaling at the point of Syk phosphorylation. Remarkably FcRL4 entered endosomes after treatment of B cells with the TLR9 agonist CpG and enhanced TLR9 signaling. We suggested that FcRL4 may act as a molecular switch in B cells to dampen adaptive immune signaling and enhance innate signaling in chronic Pf exposure. Over the last year we initiated studies to determine the Ab repertoire of conventional FcRL4- and atypical FcRL4+ MBCs. Our approach is to carry out cloning of VH and VL genes from single B cell from atypical FcRL4+ and conventional FcRL4- MBCs from Malian children and adults. We will also carry out deep sequencing of plasma blast that are greatly expanded in the peripheral blood approximately seven days after a case of malaria and are enriched in Pf-specific cells. We have thus far acquired the technology to clone VH and VL from single B cells and have obtained sequences from both Malian children and adults. To explore the interactions between Pf and the human host, we recently carried out an extensive yeast two hybrid screen between 150 Pf proteins anticipated to be exposed to the human immune system and 12,000 human open reading frames. We identified several potential interactions and have made significant progress on one. We showed that the 33kD fragment of Pf merozoite surface protein 1 (MSP133), an abundant merozoite surface protein that is shed during red blood cell invasion, binds to S100P, a member of S100 family, several of which are pro-inflammatory, damage associated molecular pattern proteins. MSP133 binds selectively to S100P with high-affinity and blocks S100P-induced NFkappaB activation in monocytes and chemotaxis and superoxide production in neutrophiles. Remarkably, S100P binds to both dimorphic alleles of MSP1, estimated to have diverged over 27 million years ago, suggesting an ancient conserved relationship between these parasite and host proteins. We postulate that MSP133 is released by Pf merozoites to attenuate potentially damaging inflammatory responses for the benefit of both the parasite and the host. S100P is expressed at highest levels in the placenta consequently we are characterizing S100P expression and localization in placentas from women who have placenta malaria and from women free of Pf infection during pregnancy. Over the last year we also explored the relationship between Pf infections and autoimmune disease. In collaboration with Dr. Silvia Bolland, we provided evidence that a genetic susceptibility to systemic lupus erythematosus (SLE) protects against cerebral malaria. Protection appears to be by immune mechanisms that allow SLE-prone mice to better control their overall inflammatory responses to parasite infections. We are further exploring the impact of Plasmodium infections on the outcome of autoimmune disease in mice. In addition, in collaboration with Dr. Inaki Sanz (Emory University) we are characterizing our Malian cohort for autoantibodies associated with SLE. Our studies of severe cerebral malaria in mice has led us to better characterize the sequence of events that contribute to the disease. To do so we have established a collaboration with Dr. Dorian McGavern to use two photon, intravital imaging to view the cellular events that occur following infection with a mouse Plasmodium that results in cerebral disease using fluorescently labeled parasites, T cell, mononuclear cells and markers for vascular permeability.